Research ArticleArsenic-Redox Transformation andPlant Growth Promotion by Purple Nonsulfur BacteriaRhodopseudomonas palustris CS2 andRhodopseudomonas faecalis SS5

Kanza Batool Fatima tuz Zahra and Yasir Rehman

Department of Microbiology amp Molecular Genetics University of the Punjab Lahore Pakistan

Correspondence should be addressed to Yasir Rehman yasirmmgpuedupk

Received 5 November 2016 Revised 9 January 2017 Accepted 19 January 2017 Published 12 March 2017

Academic Editor Wen-Jun Li

Copyright copy 2017 Kanza Batool et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Arsenic (As) is a well-known toxicmetalloid found naturally and released by different industries especially in developing countriesPurple nonsulfur bacteria (PNSB) are known for wastewater treatment and plant growth promoting abilities As-resistant PNSBwere isolated from a fish pond Based on As-resistance and plant growth promoting attributes 2 isolates CS2 and SS5 were selectedand identified as Rhodopseudomonas palustris and Rhodopseudomonas faecalis respectively through 16S rRNA gene sequencingMaximum As(V) resistance shown by R faecalis SS5 and R palustris CS2 was up to 150 and 100mM respectively R palustris CS2showed highest As(V) reduction up to 629 (629plusmn024mM) while R faecalis SS5 showedmaximumAs(III) oxidation up to 96(48 plusmn 032mM) respectively Highest auxin production was observed by R palustris CS2 and R faecalis SS up to 7718 plusmn 37 and7667 plusmn 28 120583gmLminus1 respectively Effects of these PNSB were tested on the growth of Vigna mungo plants A statistically significantincrease in growthwas observed in plants inoculatedwith isolates compared to uninoculated plants both in presence and in absenceof As R palustris CS2 treated plants showed 17 (281 plusmn 087 cm) increase in shoot length and 217 (707 plusmn 042 cm) increase inroot length whereas R faecalis SS5 treated plants showed 128 (2709plusmn081 cm) increase in shoot length and 188 (69plusmn034 cm)increase in root length as compared to the control plants In presence of As R palustris CS2 increased shoot length up to 263(210plusmn11 cm) while root length increased up to 313 (53plusmn04 cm) whereas R faecalis SS5 inoculated plants showed 25 (207 plusmn14 cm) increase in shoot length and 333 (54 plusmn 065 cm) increase in root length as compared to the control plants Bacteria withsuch diverse abilities could be ideal for plant growth promotion in As-contaminated sites

1 Introduction

Arsenic (As) is a ubiquitous toxic metalloid which is alsoreleased from several industrial processes such as miningtanning of hides and combustion of coal Due to thesereasons the level of As in drinking waters of many areas hasbeen increased beyondWHO standard drinking water limits(001mg Lminus1) [1] As is primarily found in two major formsarsenate [As(V)] and arsenite [As(III)] [2] As(III) is reportedto be sim100 times more toxic as compared to As(V) [3 4]As(V) causes harm to the cells by being analogous to phos-phorous replacing the latter in many chemical reactions [5]As(III) on the other hand affects activity of many enzymes

by reacting with their sulfhydryl groups [6] As is toxic toall life forms including plants animals and human beingsLong-term exposure toAs especially through drinkingwatercan cause arsenicosis which is manifested as different types ofcancers and skin allergies In plants the toxicity of As causesreduced growth and low yields [7] As is toxic to microorgan-isms as well however several bacteria are known to resist anddetoxifyAs As-resistant bacteria are known to have effects onAs-oxidationreduction methylation and demethylation aswell as sorption and desorption as a result of their survivalstrategies against As-toxicity [8] Such As-resistant bacteriaare ideal for the bioremediation of As-contaminated landsHowever given the possible complexity and severity of the

HindawiBioMed Research InternationalVolume 2017 Article ID 6250327 8 pageshttpsdoiorg10115520176250327

2 BioMed Research International

As-polluted sites the bacteria should bemetabolically diversein order to thrive in different environments

Purple nonsulfur bacteria (PNSB) are one of the mostmetabolically diverse and adaptable bacteria They are pho-totrophic and are widely found in nature especially in sub-merged habitats that are exposed to sunlight such as ricepaddies sewage dumping sites and riverbedsThese bacteriahave an extensive range of growth modes and are capable ofgrowing photoautotrophicallyphotoheterotrophically underanaerobic conditions in presence of incandescent light utiliz-ing a variety of organic substrates and can also grow chemo-organotrophically under aerobic dark conditions [9] Theseproperties make them ideal for wastewater bioremediation[10] Several strains of PNSB are also known to have plantgrowth promoting abilities in terms of nitrogen fixation [11]phosphate solubilization and production of phytohormonessuch as indole-3-acetic acid and 5-aminolevulinic (ALA) [12]Themost commongenera areRhodobacter andRhodopseudo-monas detected in 73 and 80 of the samples respectively[13] Some PNSB have also been reported to resist and detox-ify several toxic compounds such as As [14] ConsequentlyPNSB can also play a substantial role in As-biogeochemistryand bioremediation

Isolation and characterization of metabolically diversePNSB having abilities not only to detoxify As but also tosupport plant growth could be very useful for bioremediationpurposes This could help in reclamation of As-polluted sitesfor agriculture purposes

2 Materials and Method

21 Sampling Sampling was done from the fish pond nearthe University of the Punjab Quaid-e-Azam campus LahorePakistan (31∘29101584055410158401015840N 74∘17101584031910158401015840E) Sampling location inthe pond was so selected that it was shallow enough toallow penetration of sunlight and deep enough to haveanoxic conditions perfect for PNSB Thus 3-4 feet deepwater and sediment samples were collected Temperature andpH of the samples were recorded As(V) and As(III) werepurchased asNa

2HAsO

4sdot7H2OandNaAsO

2 respectively All

the experiments were performed in triplicate

22 Enrichment and Isolation of PNSB Media suggested byBiebl and Pfennig [15] with slight modifications was usedThemedia contained freshly prepared basalmedium (033 gmKH2PO4 033 gm MgSO

4sdot7H2O 033 gm NaCl 005 gm

CaCl2sdot2H2O 05 gm NH

4Cl 10 gm sodium succinate and

002 gm yeast extract Lminus1) to which 05mL FeSO4sdot7H2O

(002) and 10mL trace salt solution (100mg ZnSO4sdot7H2O

30mgMnSO4sdot4H2O 300mgH

3BO3 200mg CoCl

2sdot6H2O

20mg NiCl2sdot6H2O 30mg Na

2MoO4Lminus1) were added and

autoclaved After autoclaving while still warm 450mL ofmedia was aseptically poured in autoclaved 500mL Schottbottles Media were allowed to cool and 50mL of eachsample (10 inoculum water plus sediment) was inoculatedaseptically leaving little headspace Media were overlaidwith mineral oil and incubated at 28∘C in the presence ofincandescent light Once the enrichment reddish marooncolored bloom was achieved (sim10 days incubation) samples

were drawn aseptically serially diluted and plated on Pfennigagar media plates Media supplemented with different carbonsources (sodium acetate sodium citrate maleic acid sodiumsuccinate and sodium lactate) were also used to concludewhich carbon source is more suitable for the growth of PNSBPlates were incubated at 28∘C for 5ndash7 days in anaerobic jars(Oxoid) containing anaerobic sachet (Merck) in incandescentlight After incubation PNSB were purified by streak platemethod and characterized PNSB were then cultured onPfennig agar media (succinate) containing 10mM As(V)PNSB that were able to grow in presence of As(V) wereselected for further studies

23 Minimum Inhibitory Concentration (MIC) of As(V)Pfennigmedia containing 50mM 100mM or 150mMAs(V)were prepared and dispensed in screw capped tubes followedby autoclaving The media were inoculated with overnightcultures overlaid with mineral oil and incubated at 28∘C inincandescent light for 6-7 days Presence or absence of growthwas recorded

24 As-OxidationReduction Assay

241 Qualitative Assay Mineralmedium [16] supplementedwith 10mM As(V) or 5mM As(III) was inoculated withPNSB and incubated at 37∘C for 48 hours After incubation200120583L of each supernatant was taken inmicrotiter plate wellsand mixed with 001M 12 120583L KMNO

4 Color development

was observed and recorded A change from purple to yellow-ish brown color indicates As(V) reduction to As(III) [17]

242 Quantitative Assay Overnight PNSB cultures wereharvested and pellets were washed (twice) with and resus-pended in normal saline Cell suspensions were divided intotwo sets of tubes one set was supplemented with 100mMAs(V) and the other set was supplemented with 50mMAs(III) followed by incubation at 37∘C for 5 hours Sampleswere drawn at the start and after incubation Samples werecentrifuged and supernatants were analyzed for estimationof As(V) oxidationreduction as reported by Cummings etal [18] with slight modifications Standard curve was createdusing known concentrations of As(V) ranging from zero to1000mM As(V) 100 120583L of sample was mixed with 100 120583Lof 24mM HCl followed by immediate vortexing 100120583L ofthe acidified sample was mixed with 900 120583L of the coloringreagent followed by immediate vortexing The samples wereincubated at 78∘C in a water bath for 20 minutes and kept onice for 5 minutes Samples were quantified at 865 nm usingspectrophotometer (CECIL CE 7200)

25 Determination of CrossMetal Resistance Selected isolateswere tested for resistance against other metals Media con-taining 10mMCo(NO

3)2sdot6H2O ZnSO

4sdot7H2ONiCl

2sdot6H2O

CdCl2sdot5H2O K2CrO4 or CuSO

4sdot5H2O each were prepared

The media flasks were inoculated with overnight brothcultures and incubated at 37∘C for 72 hours After completionof incubation absence or presence of growth was recorded

26 16S rRNA Gene Sequencing and Phylogenetic Analysis16S rRNA gene of selected PNSB was sequenced from

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Table 1 Characteristics of As-resistant PNSB isolated in this study

Strain Gram reaction Sporestaining Catalase Oxidase Auxin

productionHCN

production Phosphate As(V)-reduction

As(III)-oxidation

CS1 Gram-negative minus Positive Positive + +++ minus +++ minus

CS2 Gram-negative minus Positive Positive + ++ minus +++ minus

CS4 Gram-negative minus Positive Positive + minus minus +++ minus

SS5 Gram-negative minus Positive Positive + +++ minus minus +++SS6 Gram-negative minus Positive Positive + minus + +++ minus

SS7 Gram negative minus Positive Positive + ++ minus minus +SS8 Gram-negative minus Positive Positive + minus minus + minus

SS9 Gram-negative minus Positive Positive + minus minus minus +

Macrogen (Korea) using primers 518F (CCAG-CAGCCGCGGTAATACG) and 800R (TACCAGGGT-ATCTAATCC) Base call quality was checked thoughFinchTV Sequences were classified using NCBI BLAST(httpsblastncbinlmnihgovBlastcgiPAGE TYPE=Blast-Search) and nearest homologues were downloaded Multiplesequence alignment was done in ClustalW Neighbour-Joining (NJ) phylogenetic tree [19] was made in MEGA50 [20] with 100 bootstrap replicates [21] as an estimate ofbranch support

27 Plant Growth Promoting Attributes of PNSB

271 Auxin Production LB-broth supplemented with500mgmLminus1 tryptophan was prepared and inoculated withovernight PNSB cultures followed by 7 days of incubation at37∘C on 150 rpm agitation Following incubation cultureswere centrifuged and 100120583L supernatant was mixed with200120583L Salkowskirsquos reagent (10mL 005M FeCl

3 50mL 35

HClO4) [22] in microtiter plate followed by incubation in

dark for 30 minutes Appearance of pinkish color indicatedauxin production Absorbance was measured at 535 nmusing Epoch BioTek plate reader

272 Phosphate Solubilization PNSB were streaked onPikovskaya [23] media followed by incubation at 28∘C for7 days Following incubation plates were observed for thepresence or absence of clear zones around PNSB colonies anindication of phosphate solubilization

273 Hydrogen Cyanide (HCN) Production PNSB wereswabbed on nutrient agar supplemented with glycine(44 gmLminus1) and a filter paper soaked in 20 Na

2CO3solu-

tion (in 05 picric acid) was placed on the top of theagar surface [24 25] After incubation at 28∘C for 4 daysdevelopment of orange to red color of the filter paperindicated the positive result for HCN production

28 Plant-Microbe Interaction Experiments PNSB were cul-tured in Pfennig broth media as described earlier Cells wereharvested and resuspended in autoclave water to achieve05600 nm optical density Vigna mungo seeds purchased from

Punjab Seed Corporation were surface-sterilized with 01

HgCl2for 20 minutes with continuous shaking followed by

washing with distilled water 3-4 times Seeds were incubatedin separate as well as in mixed PNSB suspensions (consor-tium) for 15ndash20 minutes at room temperature Followingincubation seeds were sown in 16 cm wide and 135 cm deeppots each containing equal amount of sieved soil Eightseeds were sown per pot and after germination plants werethinned to five plants per pot Pots were divided into 4 sets(a) pots containing uninoculated seeds (b) pots containinginoculated seeds (c) pots containing uninoculated seeds andAs [10mM As(V) and 01mM As(III)] and (d) the potscontaining inoculated seeds as well as As [10mM As(V) and01mM As(III)] Each set contained 10 pots The pots wereplaced in 12-hour photoperiod at 25 plusmn 2∘C for two weeksWatering was done regularly

29 Statistical Analysis Results were analyzed for statisticalsignificance using analysis of variance (ANOVA) followedby post hoc analysis using two-tailed 119905-tests (119901 = 005) inorder to compare different treatments with each other Thethreshold of significance was also adjusted using Bonferronicorrection All the biostatistical analysis was done usingMicrosoft Excel 2013 data analysis toolkit

3 Results

31 Isolation and Identification of As-Resistant PNSB Pur-plish maroon colored bloom was achieved in the enrichmentSchott bottles after incubation of 10 days at 28∘C in incan-descent light Media containing sodium acetate and sodiumlactate did not show any growthMalic acid containingmediagave sim108 times 106 CFUmLminus1 and sodium succinate containingmedia gave sim7 times 107 CFUmLminus1 whereas CFUmLminus1 onsodium citrate containing media was 2 times 107 (Table 1) All theisolated PNSB were catalase and oxidase positive All isolateswere Gram-negative and nonspore former (Table 1)

16S rRNA sequence analysis of the PNSB showed thatisolate CS2 showed maximum homology to Rhodopseu-domonas palustris (99) whereas isolate SS5 showed max-imum homology to Rhodopseudomonas faecalis (99) whenaligned through nucleotide BLAST tool of NCBI The resultswere confirmed by making NJ-phylogenetic tree IsolateCS2 grouped with R palustris whereas isolate SS5 grouped

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Table 1 Characteristics of As-resistant PNSB isolated in this study

Strain Gram reaction Sporestaining Catalase Oxidase Auxin

productionHCN

production Phosphate As(V)-reduction

As(III)-oxidation

CS1 Gram-negative minus Positive Positive + +++ minus +++ minus

CS2 Gram-negative minus Positive Positive + ++ minus +++ minus

CS4 Gram-negative minus Positive Positive + minus minus +++ minus

SS5 Gram-negative minus Positive Positive + +++ minus minus +++SS6 Gram-negative minus Positive Positive + minus + +++ minus

SS7 Gram negative minus Positive Positive + ++ minus minus +SS8 Gram-negative minus Positive Positive + minus minus + minus

SS9 Gram-negative minus Positive Positive + minus minus minus +

Macrogen (Korea) using primers 518F (CCAG-CAGCCGCGGTAATACG) and 800R (TACCAGGGT-ATCTAATCC) Base call quality was checked thoughFinchTV Sequences were classified using NCBI BLAST(httpsblastncbinlmnihgovBlastcgiPAGE TYPE=Blast-Search) and nearest homologues were downloaded Multiplesequence alignment was done in ClustalW Neighbour-Joining (NJ) phylogenetic tree [19] was made in MEGA50 [20] with 100 bootstrap replicates [21] as an estimate ofbranch support

27 Plant Growth Promoting Attributes of PNSB

271 Auxin Production LB-broth supplemented with500mgmLminus1 tryptophan was prepared and inoculated withovernight PNSB cultures followed by 7 days of incubation at37∘C on 150 rpm agitation Following incubation cultureswere centrifuged and 100120583L supernatant was mixed with200120583L Salkowskirsquos reagent (10mL 005M FeCl

3 50mL 35

HClO4) [22] in microtiter plate followed by incubation in

dark for 30 minutes Appearance of pinkish color indicatedauxin production Absorbance was measured at 535 nmusing Epoch BioTek plate reader

272 Phosphate Solubilization PNSB were streaked onPikovskaya [23] media followed by incubation at 28∘C for7 days Following incubation plates were observed for thepresence or absence of clear zones around PNSB colonies anindication of phosphate solubilization

273 Hydrogen Cyanide (HCN) Production PNSB wereswabbed on nutrient agar supplemented with glycine(44 gmLminus1) and a filter paper soaked in 20 Na

2CO3solu-

tion (in 05 picric acid) was placed on the top of theagar surface [24 25] After incubation at 28∘C for 4 daysdevelopment of orange to red color of the filter paperindicated the positive result for HCN production

28 Plant-Microbe Interaction Experiments PNSB were cul-tured in Pfennig broth media as described earlier Cells wereharvested and resuspended in autoclave water to achieve05600 nm optical density Vigna mungo seeds purchased from

Punjab Seed Corporation were surface-sterilized with 01

HgCl2for 20 minutes with continuous shaking followed by

washing with distilled water 3-4 times Seeds were incubatedin separate as well as in mixed PNSB suspensions (consor-tium) for 15ndash20 minutes at room temperature Followingincubation seeds were sown in 16 cm wide and 135 cm deeppots each containing equal amount of sieved soil Eightseeds were sown per pot and after germination plants werethinned to five plants per pot Pots were divided into 4 sets(a) pots containing uninoculated seeds (b) pots containinginoculated seeds (c) pots containing uninoculated seeds andAs [10mM As(V) and 01mM As(III)] and (d) the potscontaining inoculated seeds as well as As [10mM As(V) and01mM As(III)] Each set contained 10 pots The pots wereplaced in 12-hour photoperiod at 25 plusmn 2∘C for two weeksWatering was done regularly

29 Statistical Analysis Results were analyzed for statisticalsignificance using analysis of variance (ANOVA) followedby post hoc analysis using two-tailed 119905-tests (119901 = 005) inorder to compare different treatments with each other Thethreshold of significance was also adjusted using Bonferronicorrection All the biostatistical analysis was done usingMicrosoft Excel 2013 data analysis toolkit

3 Results

31 Isolation and Identification of As-Resistant PNSB Pur-plish maroon colored bloom was achieved in the enrichmentSchott bottles after incubation of 10 days at 28∘C in incan-descent light Media containing sodium acetate and sodiumlactate did not show any growthMalic acid containingmediagave sim108 times 106 CFUmLminus1 and sodium succinate containingmedia gave sim7 times 107 CFUmLminus1 whereas CFUmLminus1 onsodium citrate containing media was 2 times 107 (Table 1) All theisolated PNSB were catalase and oxidase positive All isolateswere Gram-negative and nonspore former (Table 1)

16S rRNA sequence analysis of the PNSB showed thatisolate CS2 showed maximum homology to Rhodopseu-domonas palustris (99) whereas isolate SS5 showed max-imum homology to Rhodopseudomonas faecalis (99) whenaligned through nucleotide BLAST tool of NCBI The resultswere confirmed by making NJ-phylogenetic tree IsolateCS2 grouped with R palustris whereas isolate SS5 grouped

4 BioMed Research International

NR 0368651 Bradyrhizobium japonicum strain 3I1b6 16S ribosomal RNA gene partial sequence

NR 0448692 Rhizobium lupini strain DSM 30140 16S ribosomal RNA gene partial sequence

NR 1373311 Bradyrhizobium subterraneum strain 58 2-1 16S ribosomal RNA partial sequence

NR 0286411 Rhodopseudomonas rhenobacensis strain Klemme Rb 16S ribosomal RNA gene complete sequence

NR 0743131 Nitrobacter hamburgensis strain X14 16S ribosomal RNA gene complete sequence

NR 1220991 Rhodopseudomonas pseudopalustris strain DSM 123 16S ribosomal RNA gene partial sequence

NR 0367711 Rhodopseudomonas pseudopalustris strain DSM 123 16S ribosomal RNA gene partial sequence

NR 0249711 Rhodopseudomonas faecalis strain gc 16S ribosomal RNA gene partial sequence

SS5

NR 1084861 Rhodopseudomonas harwoodiae strain JA531 16S ribosomal RNA gene partial sequence

NR 1155421 Rhodopseudomonas palustris strain ATCC 17001 16S ribosomal RNA gene complete sequence

CS2

NR 1085281 Rhodopseudomonas thermotolerans strain JA576 16S ribosomal RNA gene partial sequence

NR 1085271 Rhodopseudomonas pentothenatexigens strain JA575 16S ribosomal RNA gene partial sequence

100

97

100

59

44

64

100

9259

56

94

Figure 1 NJ-phylogenetic tree of PNSB isolates CS2 and SS5 along with their closest homologues from NCBI nucleotide BLASTresults Sequences were aligned with ClustalW and tree was made in MEGA 5 using 100 bootstrap replicates Isolate CS2 grouped withRhodopseudomonas palustris and SS5 grouped with Rhodopseudomonas faecalis

0

20

40

60

80

100

120

140

160

CS1 CS2 CS4 SS5 SS6 SS7 SS8 SS9

As(

V) c

once

ntra

tion

(mM

)

PNSB

Figure 2 Minimum inhibitory concentration of As(V) againstPNSB Highest concentration of As(V) was resisted byR faecalis SS5and R palustris CS2 up to 150 and 100mM respectively

with R faecalis (Figure 1) The sequences were submittedin NCBI GenBank under the accession numbers KY098792and KY098793 for R palustris CS2 and R faecalis SS5respectively

32 Heavy Metal Resistance MaximumAs(V) resistance wasshown by R faecalis SS5 up to 150mM whereas R palustrisCS2 resisted up to 100mM As(V) (Figure 2) All the isolateswere resistant to 10mM Cr Ni and Zn while they weresensitive to the Cu Cd and Co

33 Estimation of As-OxidationReduction KMNO4

wasused as reagent which changes its color to yellow in presenceof As(III) but retains its color in the presence of As(V) RpalustrisCS2was found to beAs(V) reducers whileR faecalisSS5 was found to be As(III)-oxidizer (Table 1)

Isolates R palustris CS2 showed highest As(V) reductionpotential and reduced 629 (629 plusmn 024mM) As(V) in5 hours of incubation Isolates CS4 and SS6 reduced 48(48plusmn04mM)As(V) whereas isolate SS6 reduced 42 (42plusmn031mM) As(V) Maximum As(III) oxidation was exhibitedby R faecalis SS5 and oxidized 96 (48 plusmn 032mM) As(III)in 5 hours of incubation (Figure 3)

34 Plant Growth Promoting Activities of PNSB Isolate Rfaecalis SS5 was strongly positive for the HCN productionwhile R palustris CS2 was weakly positive as indicated bythe intensity of the orange color developed on the filter paper(Table 1) All the isolates were positive for auxin productionas well and maximum auxin production was exhibited by RpalustrisCS2 (7718plusmn37 120583gmLminus1) andR faecalis SS5 (7667 plusmn28 120583gmLminus1) (Figure 4) All the isolates were negative forphosphate solubilization (Table 1)

35 Effects of PNSB on Plant Growth Different sets ofpots showed different germination rates according to theconditions provided Germination rate was affected in thepresence of As (data not shown)

Control plants were exposed neither to As nor to PNSBAverage shoot length was 240 plusmn 09 cm while average root

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0

1

2

3

4

5

6

7

CS1 CS2 CS4 SS5 SS6 SS7 SS8 SS9PNSB

As(V) reductionAs(III) oxidation

As c

once

ntra

tion

(mM

)

Figure 3 As-oxidationreduction by PNSB Bacterial cell suspen-sions were incubated for 5 hours in normal saline containing either10mMAs(V) or 5mMAs(III) Highest As(V) reduction was shownby R palustris CS2 up to 629 (629 plusmn 024mM) whereas highestAs(III) oxidation was shown by R faecalis SS5 up to 96 (48 plusmn032mM)

0

10

20

30

40

50

60

70

80

90

CS1 CS2 CS4 SS5 SS6 SS7 SS8 SS9PNSB

g m

Lminus1)

Auxi

n co

ncen

trat

ion

(

Figure 4 Auxin production by As-resistant PNSB R palustris CS2and R faecalis SS5 produced highest auxin concentrations up to7718 plusmn 37 and 7667 plusmn 28 120583gmLminus1

length was 58 plusmn 05 cm Increase in shoot and root lengthwas observed in the presence of PNSB R palustris CS2treated plants showed 17 (281 plusmn 087 cm) increase in shootlength and 217 (707 plusmn 042 cm) increase in root length ascompared to the control plants R faecalis SS5 treated plantsshowed 128 (2709 plusmn 081 cm) increase in shoot length and188 (69 plusmn 034 cm) increase in root length as comparedto the control plants Shoot length of the plants treated withmixed isolates (R palustris CS2 and R faecalis SS5) increasedup to 154 (277 plusmn 067 cm) whereas root length increasedup to 242 (72 plusmn 052 cm) as compared to the control plants(Figure 5)

Shoot and root lengths of plants exposed to As decreasedup to 307 (166 plusmn 058 cm) and 297 (408 plusmn 076 cm)respectively Plants exposed to both As(V) and PNSB showedincreased root and shoot lengths Shoot length of plants

treated with As and R palustris CS2 increased up to 263(210plusmn11 cm) while root length increased up to 313 (53plusmn04 cm) as compared to the control plants (plants exposedto As) R faecalis SS5 inoculated plants showed 25 (207 plusmn14 cm) increase in shoot length and 333 (54 plusmn 065 cm)increase in root length in presence of As as compared tocontrol plants Shoot and root lengths of plants treated withmixed isolates in presence of As increased up to 33 (221 plusmn07 cm) and 367 (55 plusmn 04 cm) respectively (Figure 6)

ANOVA of all the plant sets was significant and Bon-ferroni corrected posttest 119905-test indicated that the length ofplants exposed to As was significantly lower and the length ofplants exposed to PNSBwas significantly higher compared tocontrol plants (not exposed to As) (119901 le 005) The statisticalanalysis also confirmed that the length of plants exposed toboth As and PNSB was significantly higher as compared toplants exposed to As only (119901 le 005)

Wet and dry weight of plants exposed to neither As norPNSB was 14 plusmn 01 gm and 02 plusmn 005 gm respectively Rpalustris CS2 inoculated plants had 17 plusmn 01 gm wet weightwhile the dry weight was 03 plusmn 003 gm Wet weight of Rfaecalis SS5 treated plants was 16 plusmn 02 gm whereas the dryweight was 02 plusmn 004 gm

Wet and dry weight of plants exposed to As was 09 plusmn008 gm and 009 plusmn 001 gm respectively R palustris CS2treated plants had 125plusmn08 gm wet weight in the presence ofAs while the dry weight was 014plusmn 003 gmWet weight of Rfaecalis SS5 treated plants in presence of As was 11plusmn03 gmwhereas the dry weight was 012 plusmn 007 gm

4 Discussion

Resistance against As(V) and plant growth promoting activ-ities such as auxin and HCN production were the mainparameters used for the screening and selection of PNSBPNSB are known to utilize different carbon sources includingthose of TCA cycle as well as different fatty acids and evenfix CO

2for their growth [26 27] In the present study

As-resistant PNSB isolates were able to utilize malic acidsodium succinate or sodium citrate The isolates showingbest results were identified as R palustris CS2 and R faecalisSS5 by aligning their 16S rRNAgene sequenceswith theNCBInucleotide database using BLAST tool Rhodopseudomonasand many other PNSB belong to 120572-proteobacteria howeversome PNSB are also found in 120573-proteobacteria [28] Bothqualitative and quantitative assays of As-oxidationreductionshowed that R palustris CS2 had ability to reduce As(V) intoAs(III) whereas R faecalis SS5 had the potential to oxidizeAs(III) to As(V) Genetic determinants of As-resistance anddetoxification are found in the form of an operon called arsoperon thus the ability to resist and detoxify As is mostlylinked together [29] Bacteria are known to oxidize as well asreduce As and even some bacteria are reported to performboth functions [30 31] Thus PNSB have important roles toplay in the biogeochemical cycling of As as well

Bacteria are also known to produce different phytohor-mones that enhance plantsrsquo growth [32] IAA a type ofauxin exhibits one of the greatest plant growth promotingactivity [33] Both isolates R palustris CS2 and R faecalis

6 BioMed Research International

60

70

80

90

100

110

120

130

R palustris CS2 R faecalis SS5

Incr

ease

dec

reas

e as c

ompa

red

to co

ntro

ls (

)

lowastlowast

lowast

lowast

lowastlowast

lowastlowast

Root Shoot Root Shoot Root ShootRoot Shoot

Control

PlantＭ + s PlantＭ + ０NSB PlantＭ + ０NSB

SS5R palustris CS2 + R faecalis

Figure 5 Effects of As and PNSB on Vigna mungo growth as compared to the control plants (control = 100 plants exposed neither to Asnor PNSB) In presence of As shoot and root lengths decreased up to 307 and 297 respectively as compared to the control plants Inpresence of R palustrisCS2 shoot and root lengths increased up to 17 and 217 whereas in presence of R faecalis SS5 shoot and root lengthsincreased up to 128 and 188 respectively In presence of both R palustris CS2 and R faecalis SS5 shoot length and root length increasedup to 154 and 242 respectively lowastSignificantly different from control 119901 lt 005

100

105

110

115

120

125

130

135

140

Control

lowast

lowast

lowast

lowast

lowast

lowast

Root Shoot Root Shoot Root Shoot

Incr

ease

dec

reas

e as c

ompa

red

to A

s(V

)-tre

ated

cont

rols

()

R palustris CS2 R faecalis SS5

PlantＭ + Ｍ + ０NSB PlantＭ + Ｍ + ０NSB

R palustris CS2 + R faecalis SS5

Figure 6 Effects of As-resistant PNSB on Vigna mungo growth in presence of As compared to the control plants (control = 100 plantsexposed to As) R palustris CS2 treated plants showed 263 and 313 increase in shoot and root length respectively whereas R faecalis SS5treated plants showed 25 and 333 increase in shoot and root length respectively as compared to the control plants Shoot and root lengths ofplant treated with both R palustris CS2 and R faecalis SS5 increased up to 33 and 367 respectively lowastSignificantly different from control119901 lt 005

BioMed Research International 7

SS5 showed highest level of auxin production Hydrogencyanide is another secondary metabolite important for plantgrowth R palustris CS2 and R faecalis SS5 were also foundto produce hydrogen cyanide Plant growth promoting abilityof these isolates was checked with V mungo due to its fastgrowth rate Both R palustris CS2 and R faecalis SS5 wereable to enhance the growth of plants both in presence andin absence of As The increase in the growth of plants dueto PNSB in presence of As could be due to the reasonthat PNSB were able to oxidizereduce As present in thesoil thus decreasing the bioavailability of As to the plantsMoreover as these PNSB were also capable of producingphytohormones this added extra benefit for the growthof the plants Combined effect of R palustris CS2 and Rfaecalis SS5wasmore pronounced for root enhancementThiscould be due to the fact that mixed bacteria were able tooxidize As(III) as well as reduce As(V) keeping a redox cycleand thus further decreasing the bioavailability of As to theplants Table S1 (see Supplementary Material available onlineat httpsdoiorg10115520176250327) shows some of thestudies that report As-oxidationreduction or plant growthpromotion by PNSB As the table indicates manyRhodopseu-domonas andRhodobacter species are known to support plantgrowth by producing phytohormones Nookongbut et al [14]reported As-resistance and detoxification by different strainsof R palustris Wong et al [34] reported IAA production aswell as plant growth enhancement by R palustris Howeverliterature reporting both As-detoxification and plant growthpromotion by same PNSB strains is scarce The present studyreports this important aspect of PNSB strains which can beutilized to enhance plant growth in As-contaminated areas

5 Conclusions

Metabolically diverse bacteria such as PNSB with ability tonot only detoxify As but also support plant growth could beuseful both in bioremediation of As-contaminated lands andin supporting plant growth in such contaminated sites

Competing Interests

The authors have no conflict of interests in these results

Authorsrsquo Contributions

Kanza Batool performed all the experiments and wrote themanuscript Fatima tuz Zahra performed As-analysis YasirRehman supervised the study and edited the manuscript

Acknowledgments

University of the Punjab Lahore Pakistan is acknowledgedfor providing funds to conduct this research

References

[1] WHO Guidelines for Drinking-Water Quality Incorporating 1stand 2nd Addenda Volume 1 Recommendations World HealthOrganization Geneva Switzerland 2008

[2] R S Oremland and J F Stolz ldquoThe ecology of arsenicrdquo Sciencevol 300 no 5621 pp 939ndash944 2003

[3] National Academy of Sciences Medical and Biological Effectsof Environmental Pollutants-Arsenic National Academy of Sci-ences Washington DC USA 1977

[4] C K Jain and I Ali ldquoArsenic occurrence toxicity and specia-tion techniquesrdquoWater Research vol 34 no 17 pp 4304ndash43122000

[5] T Rezanka and K Sigler ldquoBiologically active compounds ofsemi-metalsrdquo Phytochemistry vol 69 no 3 pp 585ndash606 2008

[6] M Patra N Bhowmik B Bandopadhyay and A SharmaldquoComparison of mercury lead and arsenic with respect togenotoxic effects on plant systems and the development ofgenetic tolerancerdquoEnvironmental and Experimental Botany vol52 no 3 pp 199ndash223 2004

[7] C-X Li S-L Feng Y Shao L-N Jiang X-Y Lu andX-LHouldquoEffects of arsenic on seed germination and physiological acti-vities of wheat seedlingsrdquo Journal of Environmental Sciences vol19 no 6 pp 725ndash732 2007

[8] J F Stolz P Basu J M Santini and R S Oremland ldquoArsenicand selenium in microbial metabolismrdquo Annual Review ofMicrobiology vol 60 pp 107ndash130 2006

[9] M K Kim K-M Choi C-R Yin et al ldquoOdorous swine waste-water treatment by purple non-sulfur bacteria Rhodopseu-domonas palustris isolated from eutrophicated pondsrdquo Biotech-nology Letters vol 26 no 10 pp 819ndash822 2004

[10] K Takeno Y Yamaoka and K Sasaki ldquoTreatment of oil-con-taining sewage wastewater using immobilized photosyntheticbacteriardquoWorld Journal of Microbiology and Biotechnology vol21 no 8-9 pp 1385ndash1391 2005

[11] H Gamal-Eldin and K Elbanna ldquoField evidence for the poten-tial of Rhodobacter capsulatus as biofertilizer for flooded ricerdquoCurrent Microbiology vol 62 no 2 pp 391ndash395 2011

[12] R H Koh andH-G Song ldquoEffects of application ofRhodopseu-domonas sp on seed germination and growth of tomato underaxenic conditionsrdquo Journal of Microbiology and Biotechnologyvol 17 no 11 pp 1805ndash1810 2007

[13] G Holguin P Vazquez and Y Bashan ldquoThe role of sedimentmicroorganisms in the productivity conservation and reha-bilitation of mangrove ecosystems an overviewrdquo Biology andFertility of Soils vol 33 no 4 pp 265ndash278 2001

[14] P Nookongbut D Kantachote andMMegharaj ldquoArsenic con-tamination in areas surrounding mines and selection of poten-tial As-resistant purple nonsulfur bacteria for use in bioreme-diation based on their detoxification mechanismsrdquo Annals ofMicrobiology vol 66 no 4 pp 1419ndash1429 2016

[15] H Biebl and N Pfennig ldquoIsolation of members of the familyRhodospirillaceaerdquo inTheProkaryotes AHandbook onHabitatsIsolation and Identification of Bacteria M P Starr H Stolp HG Truper A Balows and H G Schlegel Eds pp 267ndash273Springer Berlin Germany 1981

[16] C Brunner M Wolf and R Bachofen ldquoEnrichment ofbitumen-degrading microorganismsrdquo FEMS Microbiology Let-ters vol 43 no 3 pp 337ndash344 1987

[17] T M Salmassi K Venkateswaren M Satomi K H Nealson DK Newman and J G Hering ldquoOxidation of arsenite by Agro-bacterium albertimagni AOL15 sp nov isolated from HotCreek Californiardquo Geomicrobiology Journal vol 19 no 1 pp53ndash66 2002

BioMed Research International 5

0

1

2

3

4

5

6

7

CS1 CS2 CS4 SS5 SS6 SS7 SS8 SS9PNSB

As(V) reductionAs(III) oxidation

As c

once

ntra

tion

(mM

)

Figure 3 As-oxidationreduction by PNSB Bacterial cell suspen-sions were incubated for 5 hours in normal saline containing either10mMAs(V) or 5mMAs(III) Highest As(V) reduction was shownby R palustris CS2 up to 629 (629 plusmn 024mM) whereas highestAs(III) oxidation was shown by R faecalis SS5 up to 96 (48 plusmn032mM)

0

10

20

30

40

50

60

70

80

90

CS1 CS2 CS4 SS5 SS6 SS7 SS8 SS9PNSB

g m

Lminus1)

Auxi

n co

ncen

trat

ion

(

Figure 4 Auxin production by As-resistant PNSB R palustris CS2and R faecalis SS5 produced highest auxin concentrations up to7718 plusmn 37 and 7667 plusmn 28 120583gmLminus1

length was 58 plusmn 05 cm Increase in shoot and root lengthwas observed in the presence of PNSB R palustris CS2treated plants showed 17 (281 plusmn 087 cm) increase in shootlength and 217 (707 plusmn 042 cm) increase in root length ascompared to the control plants R faecalis SS5 treated plantsshowed 128 (2709 plusmn 081 cm) increase in shoot length and188 (69 plusmn 034 cm) increase in root length as comparedto the control plants Shoot length of the plants treated withmixed isolates (R palustris CS2 and R faecalis SS5) increasedup to 154 (277 plusmn 067 cm) whereas root length increasedup to 242 (72 plusmn 052 cm) as compared to the control plants(Figure 5)

Shoot and root lengths of plants exposed to As decreasedup to 307 (166 plusmn 058 cm) and 297 (408 plusmn 076 cm)respectively Plants exposed to both As(V) and PNSB showedincreased root and shoot lengths Shoot length of plants

treated with As and R palustris CS2 increased up to 263(210plusmn11 cm) while root length increased up to 313 (53plusmn04 cm) as compared to the control plants (plants exposedto As) R faecalis SS5 inoculated plants showed 25 (207 plusmn14 cm) increase in shoot length and 333 (54 plusmn 065 cm)increase in root length in presence of As as compared tocontrol plants Shoot and root lengths of plants treated withmixed isolates in presence of As increased up to 33 (221 plusmn07 cm) and 367 (55 plusmn 04 cm) respectively (Figure 6)

ANOVA of all the plant sets was significant and Bon-ferroni corrected posttest 119905-test indicated that the length ofplants exposed to As was significantly lower and the length ofplants exposed to PNSBwas significantly higher compared tocontrol plants (not exposed to As) (119901 le 005) The statisticalanalysis also confirmed that the length of plants exposed toboth As and PNSB was significantly higher as compared toplants exposed to As only (119901 le 005)

Wet and dry weight of plants exposed to neither As norPNSB was 14 plusmn 01 gm and 02 plusmn 005 gm respectively Rpalustris CS2 inoculated plants had 17 plusmn 01 gm wet weightwhile the dry weight was 03 plusmn 003 gm Wet weight of Rfaecalis SS5 treated plants was 16 plusmn 02 gm whereas the dryweight was 02 plusmn 004 gm

Wet and dry weight of plants exposed to As was 09 plusmn008 gm and 009 plusmn 001 gm respectively R palustris CS2treated plants had 125plusmn08 gm wet weight in the presence ofAs while the dry weight was 014plusmn 003 gmWet weight of Rfaecalis SS5 treated plants in presence of As was 11plusmn03 gmwhereas the dry weight was 012 plusmn 007 gm

4 Discussion

Resistance against As(V) and plant growth promoting activ-ities such as auxin and HCN production were the mainparameters used for the screening and selection of PNSBPNSB are known to utilize different carbon sources includingthose of TCA cycle as well as different fatty acids and evenfix CO

2for their growth [26 27] In the present study

As-resistant PNSB isolates were able to utilize malic acidsodium succinate or sodium citrate The isolates showingbest results were identified as R palustris CS2 and R faecalisSS5 by aligning their 16S rRNAgene sequenceswith theNCBInucleotide database using BLAST tool Rhodopseudomonasand many other PNSB belong to 120572-proteobacteria howeversome PNSB are also found in 120573-proteobacteria [28] Bothqualitative and quantitative assays of As-oxidationreductionshowed that R palustris CS2 had ability to reduce As(V) intoAs(III) whereas R faecalis SS5 had the potential to oxidizeAs(III) to As(V) Genetic determinants of As-resistance anddetoxification are found in the form of an operon called arsoperon thus the ability to resist and detoxify As is mostlylinked together [29] Bacteria are known to oxidize as well asreduce As and even some bacteria are reported to performboth functions [30 31] Thus PNSB have important roles toplay in the biogeochemical cycling of As as well

Bacteria are also known to produce different phytohor-mones that enhance plantsrsquo growth [32] IAA a type ofauxin exhibits one of the greatest plant growth promotingactivity [33] Both isolates R palustris CS2 and R faecalis

6 BioMed Research International

60

70

80

90

100

110

120

130

R palustris CS2 R faecalis SS5

Incr

ease

dec

reas

e as c

ompa

red

to co

ntro

ls (

)

lowastlowast

lowast

lowast

lowastlowast

lowastlowast

Root Shoot Root Shoot Root ShootRoot Shoot

Control

PlantＭ + s PlantＭ + ０NSB PlantＭ + ０NSB

SS5R palustris CS2 + R faecalis

Figure 5 Effects of As and PNSB on Vigna mungo growth as compared to the control plants (control = 100 plants exposed neither to Asnor PNSB) In presence of As shoot and root lengths decreased up to 307 and 297 respectively as compared to the control plants Inpresence of R palustrisCS2 shoot and root lengths increased up to 17 and 217 whereas in presence of R faecalis SS5 shoot and root lengthsincreased up to 128 and 188 respectively In presence of both R palustris CS2 and R faecalis SS5 shoot length and root length increasedup to 154 and 242 respectively lowastSignificantly different from control 119901 lt 005

100

105

110

115

120

125

130

135

140

Control

lowast

lowast

lowast

lowast

lowast

lowast

Root Shoot Root Shoot Root Shoot

Incr

ease

dec

reas

e as c

ompa

red

to A

s(V

)-tre

ated

cont

rols

()

R palustris CS2 R faecalis SS5

PlantＭ + Ｍ + ０NSB PlantＭ + Ｍ + ０NSB

R palustris CS2 + R faecalis SS5

Figure 6 Effects of As-resistant PNSB on Vigna mungo growth in presence of As compared to the control plants (control = 100 plantsexposed to As) R palustris CS2 treated plants showed 263 and 313 increase in shoot and root length respectively whereas R faecalis SS5treated plants showed 25 and 333 increase in shoot and root length respectively as compared to the control plants Shoot and root lengths ofplant treated with both R palustris CS2 and R faecalis SS5 increased up to 33 and 367 respectively lowastSignificantly different from control119901 lt 005

BioMed Research International 7

SS5 showed highest level of auxin production Hydrogencyanide is another secondary metabolite important for plantgrowth R palustris CS2 and R faecalis SS5 were also foundto produce hydrogen cyanide Plant growth promoting abilityof these isolates was checked with V mungo due to its fastgrowth rate Both R palustris CS2 and R faecalis SS5 wereable to enhance the growth of plants both in presence andin absence of As The increase in the growth of plants dueto PNSB in presence of As could be due to the reasonthat PNSB were able to oxidizereduce As present in thesoil thus decreasing the bioavailability of As to the plantsMoreover as these PNSB were also capable of producingphytohormones this added extra benefit for the growthof the plants Combined effect of R palustris CS2 and Rfaecalis SS5wasmore pronounced for root enhancementThiscould be due to the fact that mixed bacteria were able tooxidize As(III) as well as reduce As(V) keeping a redox cycleand thus further decreasing the bioavailability of As to theplants Table S1 (see Supplementary Material available onlineat httpsdoiorg10115520176250327) shows some of thestudies that report As-oxidationreduction or plant growthpromotion by PNSB As the table indicates manyRhodopseu-domonas andRhodobacter species are known to support plantgrowth by producing phytohormones Nookongbut et al [14]reported As-resistance and detoxification by different strainsof R palustris Wong et al [34] reported IAA production aswell as plant growth enhancement by R palustris Howeverliterature reporting both As-detoxification and plant growthpromotion by same PNSB strains is scarce The present studyreports this important aspect of PNSB strains which can beutilized to enhance plant growth in As-contaminated areas

5 Conclusions

Metabolically diverse bacteria such as PNSB with ability tonot only detoxify As but also support plant growth could beuseful both in bioremediation of As-contaminated lands andin supporting plant growth in such contaminated sites

Competing Interests

The authors have no conflict of interests in these results

Authorsrsquo Contributions

Kanza Batool performed all the experiments and wrote themanuscript Fatima tuz Zahra performed As-analysis YasirRehman supervised the study and edited the manuscript

Acknowledgments

University of the Punjab Lahore Pakistan is acknowledgedfor providing funds to conduct this research

References

[1] WHO Guidelines for Drinking-Water Quality Incorporating 1stand 2nd Addenda Volume 1 Recommendations World HealthOrganization Geneva Switzerland 2008

[2] R S Oremland and J F Stolz ldquoThe ecology of arsenicrdquo Sciencevol 300 no 5621 pp 939ndash944 2003

[3] National Academy of Sciences Medical and Biological Effectsof Environmental Pollutants-Arsenic National Academy of Sci-ences Washington DC USA 1977

[4] C K Jain and I Ali ldquoArsenic occurrence toxicity and specia-tion techniquesrdquoWater Research vol 34 no 17 pp 4304ndash43122000

[5] T Rezanka and K Sigler ldquoBiologically active compounds ofsemi-metalsrdquo Phytochemistry vol 69 no 3 pp 585ndash606 2008

[6] M Patra N Bhowmik B Bandopadhyay and A SharmaldquoComparison of mercury lead and arsenic with respect togenotoxic effects on plant systems and the development ofgenetic tolerancerdquoEnvironmental and Experimental Botany vol52 no 3 pp 199ndash223 2004

[7] C-X Li S-L Feng Y Shao L-N Jiang X-Y Lu andX-LHouldquoEffects of arsenic on seed germination and physiological acti-vities of wheat seedlingsrdquo Journal of Environmental Sciences vol19 no 6 pp 725ndash732 2007

[8] J F Stolz P Basu J M Santini and R S Oremland ldquoArsenicand selenium in microbial metabolismrdquo Annual Review ofMicrobiology vol 60 pp 107ndash130 2006

[9] M K Kim K-M Choi C-R Yin et al ldquoOdorous swine waste-water treatment by purple non-sulfur bacteria Rhodopseu-domonas palustris isolated from eutrophicated pondsrdquo Biotech-nology Letters vol 26 no 10 pp 819ndash822 2004

[10] K Takeno Y Yamaoka and K Sasaki ldquoTreatment of oil-con-taining sewage wastewater using immobilized photosyntheticbacteriardquoWorld Journal of Microbiology and Biotechnology vol21 no 8-9 pp 1385ndash1391 2005

[11] H Gamal-Eldin and K Elbanna ldquoField evidence for the poten-tial of Rhodobacter capsulatus as biofertilizer for flooded ricerdquoCurrent Microbiology vol 62 no 2 pp 391ndash395 2011

[12] R H Koh andH-G Song ldquoEffects of application ofRhodopseu-domonas sp on seed germination and growth of tomato underaxenic conditionsrdquo Journal of Microbiology and Biotechnologyvol 17 no 11 pp 1805ndash1810 2007

[13] G Holguin P Vazquez and Y Bashan ldquoThe role of sedimentmicroorganisms in the productivity conservation and reha-bilitation of mangrove ecosystems an overviewrdquo Biology andFertility of Soils vol 33 no 4 pp 265ndash278 2001

[14] P Nookongbut D Kantachote andMMegharaj ldquoArsenic con-tamination in areas surrounding mines and selection of poten-tial As-resistant purple nonsulfur bacteria for use in bioreme-diation based on their detoxification mechanismsrdquo Annals ofMicrobiology vol 66 no 4 pp 1419ndash1429 2016

[15] H Biebl and N Pfennig ldquoIsolation of members of the familyRhodospirillaceaerdquo inTheProkaryotes AHandbook onHabitatsIsolation and Identification of Bacteria M P Starr H Stolp HG Truper A Balows and H G Schlegel Eds pp 267ndash273Springer Berlin Germany 1981

[16] C Brunner M Wolf and R Bachofen ldquoEnrichment ofbitumen-degrading microorganismsrdquo FEMS Microbiology Let-ters vol 43 no 3 pp 337ndash344 1987

[17] T M Salmassi K Venkateswaren M Satomi K H Nealson DK Newman and J G Hering ldquoOxidation of arsenite by Agro-bacterium albertimagni AOL15 sp nov isolated from HotCreek Californiardquo Geomicrobiology Journal vol 19 no 1 pp53ndash66 2002

6 BioMed Research International

60

70

80

90

100

110

120

130

R palustris CS2 R faecalis SS5

Incr

ease

dec

reas

e as c

ompa

red

to co

ntro

ls (

)

lowastlowast

lowast

lowast

lowastlowast

lowastlowast

Root Shoot Root Shoot Root ShootRoot Shoot

Control

PlantＭ + s PlantＭ + ０NSB PlantＭ + ０NSB

SS5R palustris CS2 + R faecalis

Figure 5 Effects of As and PNSB on Vigna mungo growth as compared to the control plants (control = 100 plants exposed neither to Asnor PNSB) In presence of As shoot and root lengths decreased up to 307 and 297 respectively as compared to the control plants Inpresence of R palustrisCS2 shoot and root lengths increased up to 17 and 217 whereas in presence of R faecalis SS5 shoot and root lengthsincreased up to 128 and 188 respectively In presence of both R palustris CS2 and R faecalis SS5 shoot length and root length increasedup to 154 and 242 respectively lowastSignificantly different from control 119901 lt 005

100

105

110

115

120

125

130

135

140

Control

lowast

lowast

lowast

lowast

lowast

lowast

Root Shoot Root Shoot Root Shoot

Incr

ease

dec

reas

e as c

ompa

red

to A

s(V

)-tre

ated

cont

rols

()

R palustris CS2 R faecalis SS5

PlantＭ + Ｍ + ０NSB PlantＭ + Ｍ + ０NSB

R palustris CS2 + R faecalis SS5

Figure 6 Effects of As-resistant PNSB on Vigna mungo growth in presence of As compared to the control plants (control = 100 plantsexposed to As) R palustris CS2 treated plants showed 263 and 313 increase in shoot and root length respectively whereas R faecalis SS5treated plants showed 25 and 333 increase in shoot and root length respectively as compared to the control plants Shoot and root lengths ofplant treated with both R palustris CS2 and R faecalis SS5 increased up to 33 and 367 respectively lowastSignificantly different from control119901 lt 005

BioMed Research International 7

SS5 showed highest level of auxin production Hydrogencyanide is another secondary metabolite important for plantgrowth R palustris CS2 and R faecalis SS5 were also foundto produce hydrogen cyanide Plant growth promoting abilityof these isolates was checked with V mungo due to its fastgrowth rate Both R palustris CS2 and R faecalis SS5 wereable to enhance the growth of plants both in presence andin absence of As The increase in the growth of plants dueto PNSB in presence of As could be due to the reasonthat PNSB were able to oxidizereduce As present in thesoil thus decreasing the bioavailability of As to the plantsMoreover as these PNSB were also capable of producingphytohormones this added extra benefit for the growthof the plants Combined effect of R palustris CS2 and Rfaecalis SS5wasmore pronounced for root enhancementThiscould be due to the fact that mixed bacteria were able tooxidize As(III) as well as reduce As(V) keeping a redox cycleand thus further decreasing the bioavailability of As to theplants Table S1 (see Supplementary Material available onlineat httpsdoiorg10115520176250327) shows some of thestudies that report As-oxidationreduction or plant growthpromotion by PNSB As the table indicates manyRhodopseu-domonas andRhodobacter species are known to support plantgrowth by producing phytohormones Nookongbut et al [14]reported As-resistance and detoxification by different strainsof R palustris Wong et al [34] reported IAA production aswell as plant growth enhancement by R palustris Howeverliterature reporting both As-detoxification and plant growthpromotion by same PNSB strains is scarce The present studyreports this important aspect of PNSB strains which can beutilized to enhance plant growth in As-contaminated areas

5 Conclusions

Metabolically diverse bacteria such as PNSB with ability tonot only detoxify As but also support plant growth could beuseful both in bioremediation of As-contaminated lands andin supporting plant growth in such contaminated sites

Competing Interests

The authors have no conflict of interests in these results

Authorsrsquo Contributions

Kanza Batool performed all the experiments and wrote themanuscript Fatima tuz Zahra performed As-analysis YasirRehman supervised the study and edited the manuscript

Acknowledgments

University of the Punjab Lahore Pakistan is acknowledgedfor providing funds to conduct this research

References

[1] WHO Guidelines for Drinking-Water Quality Incorporating 1stand 2nd Addenda Volume 1 Recommendations World HealthOrganization Geneva Switzerland 2008

[2] R S Oremland and J F Stolz ldquoThe ecology of arsenicrdquo Sciencevol 300 no 5621 pp 939ndash944 2003

[3] National Academy of Sciences Medical and Biological Effectsof Environmental Pollutants-Arsenic National Academy of Sci-ences Washington DC USA 1977

[4] C K Jain and I Ali ldquoArsenic occurrence toxicity and specia-tion techniquesrdquoWater Research vol 34 no 17 pp 4304ndash43122000

[5] T Rezanka and K Sigler ldquoBiologically active compounds ofsemi-metalsrdquo Phytochemistry vol 69 no 3 pp 585ndash606 2008

[6] M Patra N Bhowmik B Bandopadhyay and A SharmaldquoComparison of mercury lead and arsenic with respect togenotoxic effects on plant systems and the development ofgenetic tolerancerdquoEnvironmental and Experimental Botany vol52 no 3 pp 199ndash223 2004

[7] C-X Li S-L Feng Y Shao L-N Jiang X-Y Lu andX-LHouldquoEffects of arsenic on seed germination and physiological acti-vities of wheat seedlingsrdquo Journal of Environmental Sciences vol19 no 6 pp 725ndash732 2007

[8] J F Stolz P Basu J M Santini and R S Oremland ldquoArsenicand selenium in microbial metabolismrdquo Annual Review ofMicrobiology vol 60 pp 107ndash130 2006

[9] M K Kim K-M Choi C-R Yin et al ldquoOdorous swine waste-water treatment by purple non-sulfur bacteria Rhodopseu-domonas palustris isolated from eutrophicated pondsrdquo Biotech-nology Letters vol 26 no 10 pp 819ndash822 2004

[10] K Takeno Y Yamaoka and K Sasaki ldquoTreatment of oil-con-taining sewage wastewater using immobilized photosyntheticbacteriardquoWorld Journal of Microbiology and Biotechnology vol21 no 8-9 pp 1385ndash1391 2005

[11] H Gamal-Eldin and K Elbanna ldquoField evidence for the poten-tial of Rhodobacter capsulatus as biofertilizer for flooded ricerdquoCurrent Microbiology vol 62 no 2 pp 391ndash395 2011

[12] R H Koh andH-G Song ldquoEffects of application ofRhodopseu-domonas sp on seed germination and growth of tomato underaxenic conditionsrdquo Journal of Microbiology and Biotechnologyvol 17 no 11 pp 1805ndash1810 2007

[13] G Holguin P Vazquez and Y Bashan ldquoThe role of sedimentmicroorganisms in the productivity conservation and reha-bilitation of mangrove ecosystems an overviewrdquo Biology andFertility of Soils vol 33 no 4 pp 265ndash278 2001

[14] P Nookongbut D Kantachote andMMegharaj ldquoArsenic con-tamination in areas surrounding mines and selection of poten-tial As-resistant purple nonsulfur bacteria for use in bioreme-diation based on their detoxification mechanismsrdquo Annals ofMicrobiology vol 66 no 4 pp 1419ndash1429 2016

[15] H Biebl and N Pfennig ldquoIsolation of members of the familyRhodospirillaceaerdquo inTheProkaryotes AHandbook onHabitatsIsolation and Identification of Bacteria M P Starr H Stolp HG Truper A Balows and H G Schlegel Eds pp 267ndash273Springer Berlin Germany 1981

[16] C Brunner M Wolf and R Bachofen ldquoEnrichment ofbitumen-degrading microorganismsrdquo FEMS Microbiology Let-ters vol 43 no 3 pp 337ndash344 1987

[17] T M Salmassi K Venkateswaren M Satomi K H Nealson DK Newman and J G Hering ldquoOxidation of arsenite by Agro-bacterium albertimagni AOL15 sp nov isolated from HotCreek Californiardquo Geomicrobiology Journal vol 19 no 1 pp53ndash66 2002

BioMed Research International 7

SS5 showed highest level of auxin production Hydrogencyanide is another secondary metabolite important for plantgrowth R palustris CS2 and R faecalis SS5 were also foundto produce hydrogen cyanide Plant growth promoting abilityof these isolates was checked with V mungo due to its fastgrowth rate Both R palustris CS2 and R faecalis SS5 wereable to enhance the growth of plants both in presence andin absence of As The increase in the growth of plants dueto PNSB in presence of As could be due to the reasonthat PNSB were able to oxidizereduce As present in thesoil thus decreasing the bioavailability of As to the plantsMoreover as these PNSB were also capable of producingphytohormones this added extra benefit for the growthof the plants Combined effect of R palustris CS2 and Rfaecalis SS5wasmore pronounced for root enhancementThiscould be due to the fact that mixed bacteria were able tooxidize As(III) as well as reduce As(V) keeping a redox cycleand thus further decreasing the bioavailability of As to theplants Table S1 (see Supplementary Material available onlineat httpsdoiorg10115520176250327) shows some of thestudies that report As-oxidationreduction or plant growthpromotion by PNSB As the table indicates manyRhodopseu-domonas andRhodobacter species are known to support plantgrowth by producing phytohormones Nookongbut et al [14]reported As-resistance and detoxification by different strainsof R palustris Wong et al [34] reported IAA production aswell as plant growth enhancement by R palustris Howeverliterature reporting both As-detoxification and plant growthpromotion by same PNSB strains is scarce The present studyreports this important aspect of PNSB strains which can beutilized to enhance plant growth in As-contaminated areas

5 Conclusions

Metabolically diverse bacteria such as PNSB with ability tonot only detoxify As but also support plant growth could beuseful both in bioremediation of As-contaminated lands andin supporting plant growth in such contaminated sites

Competing Interests

The authors have no conflict of interests in these results

Authorsrsquo Contributions

Kanza Batool performed all the experiments and wrote themanuscript Fatima tuz Zahra performed As-analysis YasirRehman supervised the study and edited the manuscript

Acknowledgments

University of the Punjab Lahore Pakistan is acknowledgedfor providing funds to conduct this research

References

[1] WHO Guidelines for Drinking-Water Quality Incorporating 1stand 2nd Addenda Volume 1 Recommendations World HealthOrganization Geneva Switzerland 2008

[2] R S Oremland and J F Stolz ldquoThe ecology of arsenicrdquo Sciencevol 300 no 5621 pp 939ndash944 2003

[3] National Academy of Sciences Medical and Biological Effectsof Environmental Pollutants-Arsenic National Academy of Sci-ences Washington DC USA 1977

[4] C K Jain and I Ali ldquoArsenic occurrence toxicity and specia-tion techniquesrdquoWater Research vol 34 no 17 pp 4304ndash43122000

[5] T Rezanka and K Sigler ldquoBiologically active compounds ofsemi-metalsrdquo Phytochemistry vol 69 no 3 pp 585ndash606 2008

[6] M Patra N Bhowmik B Bandopadhyay and A SharmaldquoComparison of mercury lead and arsenic with respect togenotoxic effects on plant systems and the development ofgenetic tolerancerdquoEnvironmental and Experimental Botany vol52 no 3 pp 199ndash223 2004

[7] C-X Li S-L Feng Y Shao L-N Jiang X-Y Lu andX-LHouldquoEffects of arsenic on seed germination and physiological acti-vities of wheat seedlingsrdquo Journal of Environmental Sciences vol19 no 6 pp 725ndash732 2007

[8] J F Stolz P Basu J M Santini and R S Oremland ldquoArsenicand selenium in microbial metabolismrdquo Annual Review ofMicrobiology vol 60 pp 107ndash130 2006

[9] M K Kim K-M Choi C-R Yin et al ldquoOdorous swine waste-water treatment by purple non-sulfur bacteria Rhodopseu-domonas palustris isolated from eutrophicated pondsrdquo Biotech-nology Letters vol 26 no 10 pp 819ndash822 2004

[10] K Takeno Y Yamaoka and K Sasaki ldquoTreatment of oil-con-taining sewage wastewater using immobilized photosyntheticbacteriardquoWorld Journal of Microbiology and Biotechnology vol21 no 8-9 pp 1385ndash1391 2005

[11] H Gamal-Eldin and K Elbanna ldquoField evidence for the poten-tial of Rhodobacter capsulatus as biofertilizer for flooded ricerdquoCurrent Microbiology vol 62 no 2 pp 391ndash395 2011

[12] R H Koh andH-G Song ldquoEffects of application ofRhodopseu-domonas sp on seed germination and growth of tomato underaxenic conditionsrdquo Journal of Microbiology and Biotechnologyvol 17 no 11 pp 1805ndash1810 2007

[13] G Holguin P Vazquez and Y Bashan ldquoThe role of sedimentmicroorganisms in the productivity conservation and reha-bilitation of mangrove ecosystems an overviewrdquo Biology andFertility of Soils vol 33 no 4 pp 265ndash278 2001

[14] P Nookongbut D Kantachote andMMegharaj ldquoArsenic con-tamination in areas surrounding mines and selection of poten-tial As-resistant purple nonsulfur bacteria for use in bioreme-diation based on their detoxification mechanismsrdquo Annals ofMicrobiology vol 66 no 4 pp 1419ndash1429 2016

[15] H Biebl and N Pfennig ldquoIsolation of members of the familyRhodospirillaceaerdquo inTheProkaryotes AHandbook onHabitatsIsolation and Identification of Bacteria M P Starr H Stolp HG Truper A Balows and H G Schlegel Eds pp 267ndash273Springer Berlin Germany 1981

[16] C Brunner M Wolf and R Bachofen ldquoEnrichment ofbitumen-degrading microorganismsrdquo FEMS Microbiology Let-ters vol 43 no 3 pp 337ndash344 1987

[17] T M Salmassi K Venkateswaren M Satomi K H Nealson DK Newman and J G Hering ldquoOxidation of arsenite by Agro-bacterium albertimagni AOL15 sp nov isolated from HotCreek Californiardquo Geomicrobiology Journal vol 19 no 1 pp53ndash66 2002

8 BioMed Research International

[18] D E Cummings F Caccavo Jr S Fendorf and R F Rosen-zweig ldquoArsenic mobilization by the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrYrdquo Environmental Sci-ence amp Technology vol 33 no 5 pp 723ndash729 1999

[19] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[20] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[21] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the Bootstraprdquo Evolution vol 39 no 4 pp 783ndash791 1985

[22] S A Gordon andR PWeber ldquoColorimetric estimation of indo-leacetic acidrdquo Plant Physiology vol 26 no 1 pp 192ndash195 1951

[23] R E Pikovskaya ldquoMobilization of phosphorus in soil in con-nection with vital activity of some microbial speciesrdquo Mikrobi-ologiya vol 17 pp 362ndash370 1948

[24] A P G C Marques C Pires H Moreira A O S S Rangeland P M L Castro ldquoAssessment of the plant growth promotionabilities of six bacterial isolates using Zea mays as indicatorplantrdquo Soil Biology and Biochemistry vol 42 no 8 pp 1229ndash1235 2010

[25] H Lorck ldquoProduction of hydrocyanic acid by bacteriardquo Physi-ologia Plantarum vol 1 no 2 pp 142ndash146 1948

[26] F R Tabita ldquoThe biochemistry andmetabolic regulation of car-bonmetabolism andCO

2fixation in purple bacteriardquo inAnoxy-

genic Photosynthetic Bacteria R E BlankenshipM TMadiganand C E Bauer Eds pp 885ndash914 Springer Dordrecht theNetherlands 1995

[27] Y-K Oh E-H Seol M-S Kim and S Park ldquoPhotoproduc-tion of hydrogen from acetate by a chemoheterotrophic bac-terium Rhodopseudomonas palustris P4rdquo International Journalof Hydrogen Energy vol 29 no 11 pp 1115ndash1121 2004

[28] J A Lake J Larsen B Sarna et al ldquoRings reconcile genotypicand phenotypic evolution within the Proteobacteriardquo GenomeBiology and Evolution vol 7 no 12 pp 3434ndash3442 2015

[29] S Silver and L T Phung ldquoGenes and enzymes involved in bac-terial oxidation and reduction of inorganic arsenicrdquoApplied andEnvironmental Microbiology vol 71 no 2 pp 599ndash608 2005

[30] H Fan C Su Y Wang et al ldquoSedimentary arsenite-oxidizingand arsenate-reducing bacteria associated with high arsenicgroundwater from Shanyin Northwestern Chinardquo Journal ofApplied Microbiology vol 105 no 2 pp 529ndash539 2008

[31] M Shafique A Jawaid and Y Rehman ldquoAs(V) reductionAs(III) oxidation and Cr(VI) reduction by multi-metal-resist-ant Bacillus subtilis Bacillus safensis and Bacillus cereus speciesisolated from wastewater treatment plantrdquo GeomicrobiologyJournal pp 1ndash8 2016

[32] F Ahmad I Ahmad and M S Khan ldquoScreening of free-livingrhizospheric bacteria for their multiple plant growth promotingactivitiesrdquo Microbiological Research vol 163 no 2 pp 173ndash1812008

[33] E A Tsavkelova S Y Klimova T A Cherdyntseva and A INetrusov ldquoMicrobial producers of plant growth stimulators andtheir practical use a reviewrdquo Applied Biochemistry and Micro-biology vol 42 no 2 pp 117ndash126 2006

[34] W-TWong C-H Tseng S-H Hsu et al ldquoPromoting effects ofa single Rhodopseudomonas palustris inoculant on plant growthby Brassica rapa chinensis under low fertilizer inputrdquoMicrobesand Environments vol 29 no 3 pp 303ndash313 2014

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of